CN103319610A - Novel recombinant fusion protein, preparation method and use thereof - Google Patents
Novel recombinant fusion protein, preparation method and use thereof Download PDFInfo
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Abstract
The invention relates to a novel recombinant fusion protein, a preparation method and use thereof, and particularly discloses a fusion protein. The fusion protein includes an element A in a second extracellular domain D2 of VEGFR1 (vascular endothelial growth factor receptor) and an immunoglobulin component B, wherein the element A and the immunoglobulin component B are connected together in series. The fusion protein disclosed by the invention has strong binding activity of VEGF. Thus, the biological activity of ligand can be effectively restrained. The invention also provides an application of a novel recombinant fusion protein drug in treatment of diseases such as tumor and the like.
Description
Technical field
The present invention relates to biomedicine field.More specifically, the present invention relates to a kind of Novel nonnatural protein and method for making thereof and purposes.
Background technology
(Angiogenesis) occurs blood vessel is the new vascularization of being induced by blood vessel endothelial cell growth factor VEGF, main vegf receptor (VEGFR1, VEGFR2) combination by VEGF and endothelial cellular membrane surface, induce the phosphorylation of VEGFR2, thereby cause a series of signal conduction, cause vascular endothelial proliferation.If (such as tumour, moist age-related macular degeneration) VEGF excessive secretion under certain morbid state, the paraplasm of just possible induction of vascular, the result of hyperplasia promotes propagation and transfer, the promotion eyes choroidal artery hyperplasia of tumour cell and causes moist macular degeneration.
For the existing widely research of the target therapeutic agent of VEGF, the target therapeutic agent for VEGF that has got the Green Light has monoclonal antibody and gene recombinant fusion protein at present.The former comprises Arastin (Avastin), Lucentis injection liquid (Lucentis), and the latter is VEGF Trap (Zaltrap, Aflibercept claim again VEGF-Trap).Arastin is used for the treatment of large bowel cancer and lung cancer, and the Lucentis injection liquid is used for the treatment of moist age-related macular degeneration, and VEGF Trap is used for the treatment of large bowel cancer and moist age-related macular degeneration.The gene recombinant fusion protein KH902 of domestic a company research and development has also finished the clinical trial for the treatment of moist age-related macular degeneration, the new drug declaration stage such as waits for.
Therefore, the recombination fusion protein medicine of exploitation new texture is significant in the research of VEGF target therapeutic agent.
Summary of the invention
The object of the present invention is to provide a kind of Novel nonnatural protein and method for making thereof and purposes.
A first aspect of the present invention provides a kind of fusion rotein, and described fusion rotein has formula Ia or the described structure of formula Ib:
C-A-B (Ia), or
C-B-A (Ib)
Wherein,
A comprises vascular endothelial growth factor receptor 1(VEGFR1) the albumen element of the second film outskirt D2, and the length of element A is 94-103 amino acid;
B is the immunoglobulin (Ig) element;
C is optional signal peptide sequence;
"-" expression connects peptide bond or the peptide linker of above-mentioned each element.
In another preference, described element A has among the SEQ ID NO.:1 aminoacid sequence (core sequence) shown in the 25-117 position and length is 94,95,96,97,98,99 or 100 amino acid.
In another preference, the length of described element A is 100 amino acid.
In another preference, the aminoacid sequence that is arranged in aminoacid sequence (core sequence) both sides shown in the SEQ ID NO.:1 25-117 position among the described element A comes from respectively the second film outskirt D2 (Domain2) both sides aminoacid sequence of natural VE GFR1.
In another preference, the aminoacid sequence of described element A is shown in 20-119 position among the SEQ ID NO.:1.
In another preference, described D2 has flanking sequence, and described flanking sequence comprises:
Be positioned at N-terminal the first flanking sequence of D2; And/or
Be positioned at the second flanking sequence of D2 carboxyl terminal.
In another preference, described the first flanking sequence is comprised of 1-5 amino-acid residue.
In another preference, described the second flanking sequence is comprised of 1-2 amino-acid residue.
In another preference, described the first and second flanking sequences are respectively from the second film outskirt D2 (25-117 position among the SEQ ID NO.:1) both sides aminoacid sequence of natural VE GFR1.
In another preference, described the first flanking sequence is SDTGR.
In another preference, described the second flanking sequence is NT.
In another preference, the Fc fragment that described element B is people's Immunoglobulin IgG1.
In another preference, the length of described peptide linker is 0-10 amino acid, preferably is 1-5 amino acid.More preferably, peptide linker is EF (120-121 position among the SEQ ID NO.:1).
In another preference, described fusion rotein also comprises signal peptide element C.
In another preference, described fusion rotein does not contain signal peptide, and structural formula is
A-B (IIIa), or
B-A (IIIb)
In the formula, A, B and "-" described as defined above.
In another preference, the aminoacid sequence of described signal peptide is shown in 1-19 position among the SEQ ID NO:1.
In another preference, the aminoacid sequence of described fusion rotein is shown in SEQ ID NO:1.
In another preference, described fusion rotein has following characteristic:
A) with the active ED50=50-200pM of combination (preferably being ED50=60-80pM) of VEGF;
B) the VEGF capable of blocking VEGFR2 phosphorylation of inducing;
C) vascularization in the VEGF capable of blocking external or body of inducing;
D) but the migration and invasion of inhibition tumor cell; With
E) plasma half-life 〉=5 day.
A second aspect of the present invention provides a kind of albumen dimer, and described dimer is formed by two described arbitrary fusion roteins of first aspect.
In another preference, described dimer has formula IIa or the described structure of formula IIb:
Wherein,
A is the albumen element that comprises VEGFR1 the second film outskirt D2, and the length of element A is 94-103 amino acid;
B is the immunoglobulin (Ig) element;
C is optional signal peptide sequence;
"-" expression connects peptide bond or the peptide linker of said elements;
" ‖ " represents disulfide linkage.
In another preference, described fusion rotein does not contain signal peptide, and structural formula is
Wherein,
A is the albumen element that comprises VEGFR1 the second film outskirt D2, and the length of element A is 94-103 amino acid;
B is the immunoglobulin (Ig) element;
"-" expression connects peptide bond or the peptide linker of said elements;
" ‖ " represents disulfide linkage.
A third aspect of the present invention provides a kind of polynucleotide of separation, described polynucleotide encoding fusion rotein claimed in claim 1.
A fourth aspect of the present invention provides a kind of carrier, and it contains the described polynucleotide of the third aspect.
A fifth aspect of the present invention provides a kind of host cell, and it contains and is integrated with the described polynucleotide of the third aspect in the described carrier of fourth aspect or the genome.
In another preference, described host cell is prokaryotic cell prokaryocyte or eukaryotic cell (such as Chinese hamster ovary celI, NS0 cell or 293 cells).
A sixth aspect of the present invention provides a kind of product protedogenous method, and it comprises step:
Under conditions suitable for the expression, cultivate the described host cell in the 5th aspect, thereby give expression to the described fusion rotein of first aspect; With
The dimer that separates described fusion rotein or formed by described fusion rotein.
A seventh aspect of the present invention provides a kind of pharmaceutical composition, and described composition comprises:
The described albumen dimer of the described fusion rotein of first aspect and/or second aspect, and
Pharmaceutically acceptable carrier.
A eighth aspect of the present invention provides the described fusion rotein of first aspect present invention and/or the dimeric purposes of the described albumen of second aspect, for the preparation of the medicine for the treatment of disease.
In another preference, described disease is selected from: tumour, moist macular degeneration or hepatic fibrosis.
In another preference, described tumour comprises: large bowel cancer, lung cancer.
In another preference, described disease is blood vessel generation relative disease.
A ninth aspect of the present invention provides a kind of method that blood vessel generation relative disease occurs or treats blood vessel that suppresses, and comprise step: the object to needs is used the described fusion rotein of first aspect.
In another preference, described fusion rotein is used with monomer and/or dimeric forms.
In another preference, described to liking the people.
Other side of the present invention is because the disclosure of this paper is apparent to those skilled in the art.In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and can making up mutually between specifically described each technical characterictic in below (eg embodiment), thus consist of new or preferred technical scheme.As space is limited, this tired stating no longer one by one.
Description of drawings
Figure 1A is the structural representation of recombination fusion protein VEGFR1D2-Fc.
Figure 1B has shown the nucleotide sequence of a kind of recombination fusion protein VEGFR1D2-Fc of the present invention.
Fig. 1 C has shown the aminoacid sequence of a kind of recombination fusion protein VEGFR1D2-Fc of the present invention.
Fig. 2 has shown VEGFR1D2-Fc protein SDS-PAGE electrophorogram, and each swimming lane is as follows: 1 is that non-reduced condition, 2 is that Marker, 3 is reductive condition.
Fig. 3 has shown that VEGFR1D2-Fc albumen is combined the active testing result with target spot VEGF.
Fig. 4 has shown the VEGFR2 phosphorylation that VEGFR1D2-Fc VEGF capable of blocking induces.
Fig. 5 has shown that the vascular endothelial cell tubulose that VEGFR1D2-Fc VEGF capable of blocking induces forms.
Fig. 6 has shown that VEGFR1D2-Fc is inhibited to vascularization in the zebra fish body, and the VEGFR1D2-Fc of various dose (4.4,14.7,44ng) is expelled in the zebra fish blood circulation quantity of blood vessel under the intestines under the record different condition.
Fig. 7 has shown rhEndostatin and the two comparison to the experiment of vascularization inhibition in the zebra fish body of lovastatin.RhEndostatin (44,100ng) and lovastatin (4ng) are expelled in the zebra fish blood circulation, the quantity of blood vessel under the intestines under the record different condition.
Fig. 8 and 9 has shown that VEGFR1D2-Fc has the activity of inhibition lung carcinoma cell (A549) growth.
Figure 10 and 11 has shown that VEGFR1D2-Fc has the activity of inhibition colorectal cancer cells (COLO-205) growth.
Figure 12 has shown the pharmacokinetics experimental result of VEGFR1D2-Fc.
Embodiment
The inventor is through extensive and deep research, be surprised to find that at VEGFR1 the second film outskirt D2 (Domain2) and increase flanking sequence, and the fusion rotein that forms after itself and the Fc of IgG1 fragment connected has very strong VEGF and is combined activity, develop thus class Novel nonnatural protein class medicine, for example a VEGFR1D2-Fc.Finished on this basis the present invention.
Take VEGFR1D2-Fc as example, it has following functions: 1) the VEGF capable of blocking VEGFR2 phosphorylation of inducing; 2) vascularization in the VEGF capable of blocking external or body of inducing; 3) but the migration and invasion of the inhibition tumor cell of dose-dependently.
In one embodiment, the inventor is by the test confirmation, and the VEGFR1D2-Fc of expression has very strong VEGF in conjunction with activity, ED50=60-80pM.Owing under some morbid state, (such as tumour, old eye macular degeneration, hepatic fibrosis), the interior VEGF secretion of body is excessive, causes the aberrant angiogenesis hyperplasia, thereby induces the disease generation or increase the weight of the state of an illness.Therefore VEGFR1D2-Fc can be in order to treat tumour, moist macular degeneration or hepatic fibrosis.
VEGFR1 and film outskirt thereof
VEGFR albumen belongs to the receptor tyrosine kinase superfamily, is a kind of film integral protein.Outer nearly 750 amino-acid residues of part of the film of VEGFR are comprised of 7 Ig structural domains similar to immunoglobulin structure.When after its respective ligand is combined, according to its corresponding acceptor property, VEGFR albumen can be induced a series of different biological functions reactions.VEGFR albumen comprises: VEGFR1 (Flt-1), VEGFR2 (KDR/FLk-1), VEGFR3 (Flt-4) or its combination.In the present invention, be preferably VEGFR1 (Flt-1), preferred natural type VEGFR1 is wild-type.
D2 of the present invention refers to VEGFR1(Flt-1) second film outskirt (Domain2).A kind of representational D2 sequence is 25-117 position among the SEQ ID NO.:1.Studies show that, the D2 of VEGFR2 (KDR/FLk-1) and VEGFR3 (Flt-4) all can not the VEGF combination, and prior art (The EMBO Journal vol.15no.18pp.4919-4927,1996) show, the polypeptide that only is made of the D2 district does not have VEGF in conjunction with activity, the fusion rotein that for example only is made of D2 district and Fc does not have VEGF and is combined active (The EMBO Journal vol.15no.18pp.4919-4927,1996).
The immunoglobulin G element
In the present invention, applicable immunoglobulin G element is not particularly limited, and can be from people or other mammiferous immunoglobulin (Ig) elements, or its mutation-ure and derivative.Preferably from the element of people's immunoglobulin (Ig).
Immunoglobulin G while comprises four subclass: IgG1, IgG2, IgG3, IgG4.The protein structure of these four subclass has very large similarity, and four zones are arranged: a variable region (VH), three constant regions (CH1, CH2, CH3).The Fc fragment is comprised of two constant regions (CH2-CH3), a disulfide linkage is wherein arranged, so that two covalently bound homodimers of Fc fragment monomer composition in the CH2 zone.Under the normal physiological conditions, the concentration of IgG is the highest with IgG1 in the human plasma, and IgG2 takes second place, and IgG3 and IgG4 concentration are lower.
A kind of preferred G element is human IgG1 Fc fragment, or its mutation-ure, derivative.
Fusion rotein and preparation thereof
In the present invention, " recombination fusion protein ", " albumen of the present invention ", " fusion rotein of the present invention " are used interchangeably, refer to have formula Ia or the described structure of Ib, namely contain the albumen element that comprises second film outskirt of VEGFR1 D2 and the fusion rotein of immunoglobulin (Ig) element (preferred Fc).A representational example is VEGFR1D2-Fc.Albumen of the present invention can be monomer or the polymer (such as dimer) that formed by monomer.In addition, should be understood that described term also comprises active fragments and the derivative of fusion rotein.
As used herein, " separation " refers to that material separates (if natural substance, primal environment namely is natural surroundings) from its primal environment.Do not have separation and purification such as the polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
As used herein, " recombination fusion protein of separation " refers to that recombination fusion protein is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purification of Recombinant fusion rotein of standard.Basically pure albumen can produce single master tape on non-reduced polyacrylamide gel.
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.
The invention still further relates to the varient of above-mentioned polynucleotide, it is encoded protein fragments, analogue and the derivative of identical aminoacid sequence with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural occurs.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing in fact the function of its coded polypeptide.
As used herein, term " primer " refer to template pairing, under the effect of archaeal dna polymerase, can synthesize take it as starting point and the general name of the Nucleotide of living alone as a widow of the DNA chain of template complementation.Primer can be natural RNA, DNA, also can be any type of natural nucleotide.Primer even can be non-natural Nucleotide such as LNA or ZNA etc.A special sequence on primer " haply " (or " basically ") and the template on chain is complementary.Primer must with template on an abundant complementation of chain could begin to extend, but the sequence of primer needn't with the sequence complete complementary of template.Such as, add the preceding paragraph sequence not complementary with template at the 5' end of 3' end and the primer of template complementation, such primer still haply with the template complementation.As long as have sufficiently long primer can with the sufficient combination of template, the primer of non-complete complementary also can form primer-template composite with template, thereby increases.
The Nucleotide full length sequence of fusion rotein of the present invention or its element (such as VEGFR1D2) or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be according to published relevant nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can obtain in large quantity relevant sequence with recombination method.This normally is cloned into carrier with it, changes cell over to again, then separates obtaining relevant sequence from the host cell after the propagation by ordinary method.
In addition, also can synthesize relevant sequence, especially fragment length more in short-term with the method for synthetic.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
The method of using round pcr DNA amplification/RNA is optimized for and obtains gene of the present invention.The primer that is used for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is such as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or fusion rotein encoding sequence, and produce method of protein of the present invention through recombinant technology.
By the recombinant DNA technology of routine, can utilize polynucleotide sequence of the present invention to can be used to express or Restruction albumen.In general following steps are arranged:
(1). with the polynucleotide (or varient) of code book invention albumen of the present invention, or transform or transduction appropriate host cell with the recombinant expression vector that contains these polynucleotide;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Method well-known to those having ordinary skill in the art can be used for make up the DNA sequences encoding that contains albumen of the present invention and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can be effectively connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, phenotypic character with the host cell that is provided for selecting transforming, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness such as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
Comprise above-mentioned suitable dna sequence dna and the suitable carrier of promotor or control sequence, can be used for transforming suitable host cell, with can marking protein.
Host cell can be prokaryotic cell prokaryocyte, such as bacterial cell; Or the eukaryotic cell such as low, such as yeast cell; Or higher eucaryotic cells, such as mammalian cell.Representative example has: intestinal bacteria, the bacterial cell of streptomyces; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, NS0, COS7 or 293 cells etc.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can in exponential growth after date results, be used CaCl
2Method is processed, and used step is well-known in this area.Another kind method is to use MgCl
2If necessary, transforming also the method for available electroporation carries out.When the host is eukaryote, can select following DNA transfection method: calcium phosphate precipitation, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, substratum used in the cultivation can be selected from various conventional mediums.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (such as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular be expressed or be secreted into to protein in the above methods can in cell or at cytolemma.If necessary, can utilize its physics, chemical separating and purifying protein by various separation methods with other characteristic.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation processes, process the combination of (salt analysis method), centrifugal, the broken bacterium of infiltration, super processing, ultracentrifugation, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods with protein precipitant.
Antibody
Among the present invention, " antibody ", " part " are used interchangeably, and refer to that albumen of the present invention is had specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " refers to that antibody capable is incorporated into respectively albumen of the present invention or its fragment.Preferably, refer to that those can be combined with albumen of the present invention or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Antibody of the present invention can be prepared by the known various technology of those skilled in that art.
The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, such as Fab' or (Fab)
2Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered scFv molecule; Or chimeric antibody.
Peptide linker
The invention provides a kind of fusion rotein, it optionally contains peptide linker.Peptide linker size and complicacy may affect the activity of albumen.Usually, peptide linker should have enough length and snappiness, enough degree of freedom is spatially arranged to bring into play its function to guarantee two albumen that connect.Avoid simultaneously forming in the peptide linker α spiral or β-pleated sheet structure etc. to the impact of the stability of fusion rotein.
The length of connection peptides is generally 0-10 amino acid, preferably 1-5 amino acid.
Pharmaceutical composition and application process
The present invention also provides a kind of composition, and it contains the fusion rotein of the present invention of significant quantity, and pharmaceutically acceptable carrier.Usually, fusion rotein of the present invention can be formulated in nontoxic, inertia with pharmaceutically acceptable aqueous carrier medium in, wherein pH is about 5-8 usually, preferably, pH is about 6-8.
As used herein, term " significant quantity " or " effective dose " refer to and can produce function or amount active and that can be accepted by people and/or animal to people and/or animal, such as 0.001-99wt%; Better 0.01-95wt%; Better, 0.1-90wt%.
As used herein, the composition of " pharmaceutically acceptable " is applicable to people and/or Mammals and without excessive bad side reaction (such as toxicity, stimulation and transformation reactions), namely has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various vehicle and thinner.
Pharmaceutical composition of the present invention contains fusion rotein of the present invention and the pharmaceutically acceptable carrier of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Usually pharmaceutical preparation should be complementary with administering mode, and pharmaceutical composition of the present invention can be made into the injection form, for example with physiological saline or contain glucose and the aqueous solution of other assistant agents is prepared by ordinary method.Described pharmaceutical composition should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity.Pharmaceutical preparation of the present invention also can be made into sustained release preparation.
The significant quantity of fusion rotein of the present invention can change with severity of the pattern of administration and disease to be treated etc.The selection of preferred significant quantity can be determined (for example by clinical trial) according to various factors by those of ordinary skills.Described factor includes but not limited to: the pharmacokinetic parameter biological example utilization ratio of fusion rotein of the present invention, metabolism, transformation period etc.; The severity of the disease that the patient will treat, patient's body weight, patient's immune state, the approach of administration etc.For tumour patient, usually, give with the about dosage of 0.5mg-5mg/kg the weight of animals (better 2mg-4mg/kg the weight of animals) when fusion rotein of the present invention every day, can obtain gratifying effect.For example, by an urgent demand for the treatment of situation, but give the dosage that several times separate every day, or dosage is reduced pari passu.
Fusion rotein of the present invention and dimer thereof or polymer mainly comprise following advantage:
1) have very strong VEGF in conjunction with active, ED50=60-80pM;
2) the VEGF capable of blocking VEGFR2 phosphorylation of inducing;
3) vascularization in the VEGF capable of blocking external or body of inducing;
4) but the migration and invasion of inhibition tumor cell.
5) VEGF capable of blocking induces hepatic fibrosis.
6) compare with existing targeted therapy recombination fusion protein for VEGF, have a molecular weight little, advantages of simple structure and simple.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, such as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber are weight percent and parts by weight.
Make up the VEGFR1D2-Fc expression vector
The VEGFR1-D2 gene coded sequence is comprised of 300 Nucleotide, shown in 58-357 position among the SEQ ID NO.:2, comprising 279 Nucleotide of D2 encoding sequence, 15 Nucleotide of upstream flanking sequence, 6 Nucleotide of downstream flanking sequence.Add 57 signal coding sequences (being 1-57 position among the SEQ ID NO.:2) that come from the mouse IgG heavy chain at its 5' end, formed 357 Nucleotide.Aminoterminal at these 357 Nucleotide adds " Kozack " sequence and " HindIII " gene clone site, add at carboxyl terminal and formed a gene fragment that contains 378 Nucleotide in " EcoRI " gene clone site.
Synthetic product (Nanjing Jin Sirui biotechnology company is synthetic) is cut (HindIII/EcoRI) through enzyme, is cloned into the pHB-Fc plasmid vector, forms the pHB-VEGFR1D2-Fc protein expression vector.The method for making of pHB-Fc plasmid vector is as follows: with pcDNA/HA-FLAG (Accession#:FJ524378) carrier for the plasmid that sets out, added human IgG1's Fc sequence in restriction endonuclease EcoRI back, added that in restriction endonuclease HindIII front human cytomegalovirus (HCMV) promotes subsequence (Accession#:X17403), promotes sub-front to add Chinese hamster glutamine synthetase gene (Accession#:X03495) at penbritin tolerance gene back, HCMV.After sequences Design is good, entrust Shanghai Jierui Biology Engineering Co., Ltd to be synthesized transformation.
Sequence SEQ ID NO:2 is the nucleotide sequence of coding recombination fusion protein, as shown in Figure 1B.Total length 1059bp, wherein 1-57bp is signal coding sequence, and 58-357bp is the VEGFR1D2 encoding sequence, and 358-363bp is the restriction enzyme site GAATTC of EcoRI, and 364-1059bp is the Fc fragment, TGA is termination codon.
Figure 1A is the structural representation of recombination fusion protein VEGFR1D2-Fc.This schematic diagram only plays the signal effect, does not represent the concrete real structure of fusion rotein of the present invention.
Sequence SEQ ID NO:1 is for the aminoacid sequence of coding recombination fusion protein, shown in Fig. 1 C.353 amino acid of total length, molecular weight is about 80kDa.Wherein the 1-19 amino acids is signal peptide, and the 20-119 position is for containing the VEGFR1D2 fragment of flanking sequence (underscore marks), and the 120-121 position is 2 amino acid of EcoRI restriction enzyme site, and the 122-353 amino acids is the Fc fragment.
The expression of VEGFR1D2-Fc
The used host cell of protein expression is the CHO-K1 cell (Cat#CCL-61) available from ATCC company.This cell is tamed into the CHO-K1 cell that can carry out suspension culture in serum free medium (EX-CELLTM302) through a series of domestication steps.
Utilize this cell, the method by electricity turns changes the pHB-VEGFR1D2-Fc plasmid over to cell.Concrete grammar is: collect the cell that is in logarithmic phase under aseptic condition, be resuspended in perfect medium after the centrifugation (1200rpm x5min), and adjust cell density to 1x10
7Cells/ml.Get the 350ul cell suspension and be transferred to 0.4cm electricity revolving cup, turn under the condition (voltage range 200 to 350V, general 260V is about time 20ms) pulse 1 time setting electricity.Add 10 – 30ug plasmid DNA to the electric revolving cup that contains cell, behind the mixing, electric revolving cup is put into electroporation gently, the punching of promoting blood circulation.Take out electric revolving cup, left standstill 5 minutes, add the 0.6ml cell culture medium, suck out behind the mixing, forward in the culture dish, put into incubator and cultivate.Check protein expression after 24-48 hour.If any protein expression, prove that gene changes over to successfully, dilute cell this moment with substratum, then be transferred in 20 96 porocyte culture plates, and every porocyte is counted 3000-5000.Cell is through a series of pressure (glutamine synthetase inhibitor) screening, finishing screen select can high expression level VEGFR1D2-Fc cell strain.
During protein production, the cell strain cell of high expression level VEGFR1D2-Fc is seeded in the cell reactor that contains 3 liters of EX-CELLTM302 substratum, cell density is 3x10
5Cells/ml, culture condition are 37 ℃, 5%CO2.Cell is the detections such as process pH, glucose, glutamine in culturing process, and add nutritive ingredient according to indices in good time.When cell density reaches 5-6x10
6During cells/ml, culture temperature is down to 33 ℃ from 37 ℃, continues to be cultured to when Cell viability reaches 60-70% and gather in the crops.The cells and supernatant of results is through ultrafiltration and concentration, and purifying is carried out in the strain of Protein A affinity chromatography.The albumen of purifying utilizes the Lowry method to carry out quantitative assay (with reference to 2010 editions Chinese Pharmacopoeias), and the protein quantification standard substance are ox blood albumin (lot number 140619-201120, Chinese drug and food is examined and determine research institute).The albumen of producing is substantially identical with theoretical value through SDS-PAGE electrophoretic analysis size, and endotoxin content is lower than standard-required.
Fig. 2 has shown VEGFR1D2-Fc protein SDS-PAGE electrophorogram.
Embodiment 3
VEGFR1D2-Fc and target spot (VEGF) detect in conjunction with active
Utilize Enzyme-linked Immunosorbent Assay to detect (ELISA) method, measure fusion rotein and target spot (VEGF) binding characteristic.Concrete steps are as follows:
With coated damping fluid CBS (Sigma-Aldrich Co., Product code:1001329288C3041-100CAP) with VEGF-165 (Cat:11066-HNAH, Sino biological Inc.) is diluted to 500ng/ml, gets 100ul and join elisa plate (Nunc
TM, Cat:442404) in, every hole 50ng.To be coated with plate and place the 4oC refrigerator overnight.First with the coated plate of 0.05%PBS-T washing once, sealed 1 hour with 3% skimmed milk room temperature again during detection.The D2-Fc albumen that dilution is good (50,25 ..., 0.0244nM) join in the coated plate every hole 100ul.After the incubated at room one hour, abandon sample, with 0.05%PBS-T washing 5 times, then add 100ul through HRP-Rabbit Anti-Human IgG Fc (one hundred difficult to understand the leading to of Luoyang of dilution (1:20000), Cat#:C030222), incubated at room one hour, washings washing 5 times, add the HRP substrate, 2N H was used later in the lucifuge colour developing in 10-20 minute
2S0
4The 0D450 value is read in the color development stopping reaction on microplate reader.
The result shows, D2-Fc has very strong VEGF in conjunction with active (as shown in Figure 3), and ED50 is (ED50=～67pM) below nmole.
VEGFR1D2-Fc can suppress VEGFR2 phosphorylation and the HUVEC cell proliferation that VEGF induces
Because VEGF is after VEGFR2 is combined, acceptor is autophosphorylation at first, then activate the signal transduction pathway of phosphatidylinositols metabolism and the protein kinase of mitogen activation, show the mitogen characteristic of VEGF, the propagation of inducing human vascular endothelial (HUVEC).Therefore the phosphorylation that suppresses the VEGFR2 that VEGF induces can suppress the propagation of vascular endothelial cell.
The concrete steps of VEGFR2 phosphorylation experiment are as follows:
(Australia Sai Ersi biotechnology (Shanghai) Co., Ltd., production code member: HUVEC-004) transferring concentration is 2 * 10 with nutrient solution with the HUVEC cell
5/ ml joins cell in the 6 porocyte culture plates, and every hole adds 4ml cell suspension, overnight incubation in the incubator.Second day discards nutrient solution, with PBS washed twice gently, then adds 4ml and contains VEGF (R﹠amp; D, Cat#293-VE/CF) and the nutrient solution of different concns VEGFR1D2-Fc albumen, hatched 13 minutes in 37 ℃ of incubators, take out 6 orifice plates, discard nutrient solution, every hole adds 150ul lysate (100mMPMSF, Protease Inhibitor cocktail, 100mM Sodium Orthovanadete solution, these three kinds of proteinase inhibitor join in the lysate by the amount of 1:100), blow and beat gently cell after reacting 2min on ice, collect suspension, the centrifugal 1.5min of 13000rpm gets supernatant.Cell pyrolysis liquid separates with 8% SDS-PAGE separation gel, then is transferred to pvdf membrane (Merck﹠amp; Co., IPVDF., Cat No:IPVH00010).Pvdf membrane cleaned in PBST 1 minute, then with confining liquid room temperature sealing 0.5 hour.With pvdf membrane low temperature (4oC) overnight incubation, clean 3 times each 10 minutes with the anti-VEGFR2 phosphorylation antibody (Cell Signaling Technology, Cat#3770S) that passes through dilution (1:750) with PBST.Pvdf membrane and two anti-(1:2000) (HRP-Goat Anti-Rabbit IgG, Luoyang one hundred is difficult to understand logical, Cat No:C030212) low temperature (4 ℃) temperature were hatched 2 hours.Clean 3 times each 10 minutes with PBST.With A liquid and the equal-volume mixing in vitro of two kinds of reagent of B liquid of ECL test kit, then be added in the front of pvdf membrane, general 2 minutes of incubation, lid layer preservative film on pvdf membrane is wiped unnecessary luminous agent, uses at last
Detect the development band in the Full-automatic chemiluminescence image analysis system.
The result shows, VEGFR1D2-Fc can significantly suppress the phosphorylation of the VEGFR2 that VEGF induces, suppresses activity and has dose-dependently (as shown in Figure 4), even also can significantly suppress the phosphorylation of VEGFR2 at minimum dose (1nM).
The concrete steps that HUVEC cell proliferation and tubulose form experiment are as follows:
The HUVEC cell is transferred concentration to 3 * 10
5/ ml joins cell in 96 well culture plates that contain Matrigel every hole 50ul.The nutrient solution that contains VEGF (20ng/ml) and different concns VEGFR1D2-Fc (1,5,10,20ug/ml) that then will prepare joins in the culture plate every hole 50ul.Culture plate places incubator to cultivate, and in different time points (0h, 2h, 4h, 6h, 8h, 24h) microscopically mug.
Result's demonstration, propagation and the tubulose of HUVEC cell in the gel entrapment culture base that VEGFR1D2-Fc can suppress the HUVEC cell form (as shown in Figure 5).
Embodiment 5
But vascularization in the VEGFR1D2-Fc dose-dependent inhibition body
Utilize blood vessel fluorescence transgenic zebrafish model, estimate testing sample VEGFR1D2-Fc to angiopoietic impact.
1) zebra fish preparation method:
Blood vessel fluorescence transgenic zebrafish embryo's breeding is carried out in the mode of natural paired cross.4～5 pairs of Adult Zebrafishs are prepared in each mating, and average every pair can be produced 200～300 embryos.At after fertilization 6 hours (being 6hpf) and 24hpf the embryo is cleared up (removing the dead embryo), and select suitable embryo according to embryo's etap.(fish culture water water quality: add the instant sea salt of 200mg in every 1L reverse osmosis water, specific conductivity is 480～510 μ S/cm to hatch the embryo with fish culture water under 28 ℃ of conditions; PH is 6.9～7.2; Hardness is 53.7～71.6mg/L CaCO
3).Because the embryo can obtain nutritive substance from the yolk sac of self, so (9dpf) do not need feeding in after fertilization 9 days.After experiment is finished, with the tricaine methylsulfonic acid zebra fish of each etap is carried out over-exposure and process, thereby with zebra fish anesthesia execution.The operation steps that anesthesia is put to death meets the code requirement that U.S. Veterinary Medical Association (AVMA) puts to death Animal Anesthesia.
2) experimental technique:
A. determine the maximum Sublethal concentration (MNLC) of testing sample
– uses microinjection instrument, and sample is expelled to (blood circulation injection) in the fluorescence transgenic zebrafish body, and every kind of testing sample arranges a plurality of different concentration, and each concentration is all processed 30 tail zebra fishs;
Three initial detecting concentration of – testing sample.
– negative control group: group of solvents;
– blank group is used for the proof solvent can be to the zebra fish harmful;
All experimental group of – (except the blank group) all contain the solvent of same concentrations;
After the – sample preparation finishes, add up the dead quantity of zebra fish of each experimental group, use the concentration-response curve of JMP statistics Software on Drawing the best, and calculate MNLC;
If – can not obtain MNLC in initial concentration test experience, will enlarge the detectable level scope of testing sample, on be limited to maxima solubility or the stoste of testing sample.
B. the quantitative evaluation sample is to angiopoietic restraining effect
– is according to the concentration destruction curve, and every kind of testing sample is chosen 3 concentration and detected (being generally maximum Sublethal concentration (MNLC), 1/3MNLC and 1/10MNLC);
Each concentration of – is all processed (blood circulation injection) 30 tail blood vessel fluorescence transgenic zebrafishes;
– positive controls: rhEndostatin and lovastatin;
– negative control group: group of solvents;
– blank group is used for the proof solvent can not produce harmful effect to zebra fish;
All experimental group of – (except the blank group) all contain the solvent of same concentrations;
After the – sample preparation finishes, every group get at random 10 tail zebra fishs at the fluorescence microscopy Microscopic observation, take pictures and preserve picture;
– carries out image analysis with image analysis software, calculates blood vessel fluorescence signal intensity (S), carries out quantitative analysis, and statistical procedures result uses
Expression;
The drug effect calculation formula of – sample anti-angiogenesis is as follows:
– carries out statistical analysis with variance analysis and Dunnett's T-check, and p＜0.05 shows to have significant difference;
– provides representative experimental patterns.
Blood vessel fluorescence transgenic zebrafish embryo is expelled to the VEGFR1D2-Fc of various dose (4.4,14.7,44ng) and positive control medicine rhEndostatin (44,100ng) and lovastatin (4ng) in the zebra fish blood circulation by microinjection instrument after fertilization 48 hours, blood vessel under 72 hours microscopic examination zebra fish intestines, and the quantity of blood vessel under the intestines under the mug, record different condition.
3) experimental result:
(1 * PBS) does not relatively have statistical significance (p〉0.05) with blank to group of solvents, illustrate that injection solvent does not affect the vascularization of zebra fish.The blood vessel inhibiting rate of 4ng lovastatin is that (dosage of 4ng is the maximum non-deadly dosage of lovastatin to (45.6 ± 2.2) %, therefore, the blood vessel inhibiting rate of this moment is the maximal percentage inhibition of lovastatin), compare with group of solvents, statistical significance (p＜0.001) is arranged.The blood vessel inhibiting rate of 44ng and 100ng rhEndostatin is respectively (9.7 ± 2.8) % and (20.1 ± 2.6) %, compares with group of solvents, and statistical significance (p＜0.05, p＜0.001) is all arranged.Two kinds of different positive control compounds all have significant restraining effect to the blood vessel of zebra fish, so this evaluation model is reliable.
Dosage is that the VEGFR1D2-Fc vascularization inhibiting rate of 4.4ng is 7.5 ± 3.5%, but compares with group of solvents, does not have statistical significance (p〉0.05); Dosage is that the VEGFR1D2-Fc vascularization inhibiting rate of 14.7ng and 44ng is respectively (15.2 ± 3.3) % and (21.4 ± 2.4) %, compare with group of solvents, statistical significance (p＜0.01 is all arranged, p＜0.001), illustrates that VEGFR1D2-Fc has significant anti-angiogenesis effect.Dosage is in the scope of 4.4ng to 44ng, and VEGFR1D2-Fc increases along with the increase of dosage the angiopoietic inhibiting rate of zebra fish, presents the VEGFR1D2-Fc dosage dependency of anti-angiogenesis.
Blood vessel generally has complete 6-8 root under the normal zebra fish intestines, if vascularization is suppressed, then number of blood vessel reduces under the complete intestines.4.4ng the blood vessel number is the 5-6 root under the complete intestines of the zebra fish of VEGFR1D2-Fc, the blood vessel number is the 3-4 root under the complete intestines of the zebra fish of 14.7ng VEGFR1D2-Fc, and the blood vessel number is the 2-3 root under the complete intestines of the zebra fish of 44ng VEGFR1D2-Fc; Illustrated qualitatively that so also VEGFR1D2-Fc has anti-angiogenesis effect (Fig. 6).
The vascularization inhibiting rate of 44ng VEGFR1D2-Fc is (21.4 ± 2.4) %, and the vascularization inhibiting rate of 44ng rhEndostatin is (9.7 ± 2.8) %; Under same dosage (44ng), the anti-angiogenesis successful of VEGFR1D2-Fc is better than rhEndostatin (p＜0.01) (Fig. 7).
But VEGFR1D2-Fc dose-dependent inhibition tumour cell
Utilize routine techniques well known to those skilled in the art, mix following component, making final concentration is 1wt% recombinant protein solution, and its prescription is as follows:
Recombinant protein 10mg
Physiological saline adds to 10ml
Regulate pH to 6.8-7.1.
Give Balb/c mouse bare subcutaneous injection 5x10
6Individual lung cancer (A549) or large bowel cancer (COLO-205) cell treat that tumor growth is to 130-170mm
3The time random packet, treatment group is the VEGFR1D2-Fc albumen of abdominal injection high (20mg/kg), low dosage (5mg/kg) respectively, twice weekly, continuous 6 administrations, negative control is the PBS injection, and positive control is Zorubicin (3mg/kg) (large bowel cancer) or Arastin (Avastin) (5mg/kg, 20mg/kg) (lung cancer).Measure gross tumor volume twice weekly.
The result shows, during high dosage the inhibiting rate of large bowel cancer and lung cancer all reached more than 90%, and the inhibiting rate to large bowel cancer still reaches (as shown in FIG. 10 and 11) more than 94% during low dosage, and the inhibiting rate of lung cancer is reached (shown in Fig. 8 and 9) more than 85%.
Embodiment 7
The pharmacokinetic analysis of VEGFR1D2-Fc
To 16 normal Balb/c mouse subcutaneous injection 50ugVEGFR1D2-Fc respectively, and respectively at 1,2,4,8,24,48,96 after the injection, and gathered blood plasma by the female vein of eye in 144 hours, detect the content of VEGFR1D2-Fc in the blood plasma with the ELISA method.The result shows, can reach at least 5 days (120 hours) (as shown in figure 12) plasma half-life of VEGFR1D2-Fc.
All quote in this application as a reference at all documents that the present invention mentions, just as each piece document is quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (11)
1. a fusion rotein is characterized in that, described fusion rotein has formula Ia or the described structure of formula Ib:
C-A-B (Ia), or
C-B-A (Ib)
Wherein,
A comprises vascular endothelial growth factor receptor 1(VEGFR1) the albumen element of the second film outskirt D2, and the length of element A is 94-103 amino acid;
B is the immunoglobulin (Ig) element;
C is optional signal peptide sequence;
"-" expression connects peptide bond or the peptide linker of above-mentioned each element.
2. fusion rotein as claimed in claim 1 is characterized in that, described D2 has flanking sequence, and described flanking sequence comprises:
Be positioned at N-terminal the first flanking sequence of D2; And/or
Be positioned at the second flanking sequence of D2 carboxyl terminal.
3. fusion rotein as claimed in claim 1 is characterized in that, described fusion rotein has following characteristic:
A) with the active ED50=50-200pM of combination of VEGF;
B) the VEGF capable of blocking VEGFR2 phosphorylation of inducing;
C) vascularization in the VEGF capable of blocking external or body of inducing;
D) but the migration and invasion of inhibition tumor cell; With
E) plasma half-life 〉=5 day.
4. an albumen dimer is characterized in that, described dimer is formed by arbitrary described fusion rotein among two claim 1-3.
5. dimer as claimed in claim 4 is characterized in that, described dimer has formula IIa or the described structure of formula IIb:
Or
Wherein,
A is the albumen element that comprises VEGFR1 the second film outskirt D2, and the length of element A is 94-103 amino acid;
B is the immunoglobulin (Ig) element;
C is optional signal peptide sequence;
"-" expression connects peptide bond or the peptide linker of said elements;
" ‖ " represents disulfide linkage.
6. the polynucleotide of a separation is characterized in that, described polynucleotide encoding fusion rotein claimed in claim 1.
7. a carrier is characterized in that, it contains polynucleotide claimed in claim 6.
8. a host cell is characterized in that, it contains and is integrated with polynucleotide claimed in claim 6 in carrier claimed in claim 7 or the genome.
9. one kind is produced protedogenous method, it is characterized in that, it comprises step:
Under conditions suitable for the expression, cultivate host cell claimed in claim 8, thereby give expression to fusion rotein claimed in claim 1; With
The dimer that separates described fusion rotein or formed by described fusion rotein.
10. a pharmaceutical composition is characterized in that, described composition comprises:
Fusion rotein claimed in claim 1 and/or albumen dimer claimed in claim 4, and
Pharmaceutically acceptable carrier.
11. fusion rotein as claimed in claim 1 and/or the dimeric purposes of albumen claimed in claim 4 is characterized in that, for the preparation of the medicine for the treatment of disease.
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